59 research outputs found
Rotational invariance of maxwell's equations
Rotational invariance of maxwell vector equation
Timed inhibition of CDC7 increases CRISPR-Cas9 mediated templated repair.
Repair of double strand DNA breaks (DSBs) can result in gene disruption or gene modification via homology directed repair (HDR) from donor DNA. Altering cellular responses to DSBs may rebalance editing outcomes towards HDR and away from other repair outcomes. Here, we utilize a pooled CRISPR screen to define host cell involvement in HDR between a Cas9 DSB and a plasmid double stranded donor DNA (dsDonor). We find that the Fanconi Anemia (FA) pathway is required for dsDonor HDR and that other genes act to repress HDR. Small molecule inhibition of one of these repressors, CDC7, by XL413 and other inhibitors increases the efficiency of HDR by up to 3.5 fold in many contexts, including primary T cells. XL413 stimulates HDR during a reversible slowing of S-phase that is unexplored for Cas9-induced HDR. We anticipate that XL413 and other such rationally developed inhibitors will be useful tools for gene modification
Cullin3-KLHL15 ubiquitin ligase mediates CtIP protein turnover to fine-tune DNA-end resection
Human CtIP is a decisive factor in DNA double-strand break repair pathway choice by enabling DNA-end resection, the first step that differentiates homologous recombination (HR) from non-homologous end-joining (NHEJ). To coordinate appropriate and timely execution of DNA-end resection, CtIP function is tightly controlled by multiple protein-protein interactions and post-translational modifications. Here, we identify the Cullin3 E3 ligase substrate adaptor Kelch-like protein 15 (KLHL15) as a new interaction partner of CtIP and show that KLHL15 promotes CtIP protein turnover via the ubiquitin-proteasome pathway. A tripeptide motif (FRY) conserved across vertebrate CtIP proteins is essential for KLHL15-binding; its mutation blocks KLHL15-dependent CtIP ubiquitination and degradation. Consequently, DNA-end resection is strongly attenuated in cells overexpressing KLHL15 but amplified in cells either expressing a CtIP-FRY mutant or lacking KLHL15, thus impacting the balance between HR and NHEJ. Collectively, our findings underline the key importance and high complexity of CtIP modulation for genome integrity
Active nuclear import and cytoplasmic retention of activation-induced deaminase
The enzyme activation-induced deaminase (AID) triggers antibody diversification in B cells by catalyzing deamination and consequently mutation of immunoglobulin genes. To minimize off-target deamination, AID is restrained by several regulatory mechanisms including nuclear exclusion, thought to be mediated exclusively by active nuclear export. Here we identify two other mechanisms involved in controlling AID subcellular localization. AID is unable to passively diffuse into the nucleus, despite its small size, and its nuclear entry requires active import mediated by a conformational nuclear localization signal. We also identify in its C terminus a determinant for AID cytoplasmic retention, which hampers diffusion to the nucleus, competes with nuclear import and is crucial for maintaining the predominantly cytoplasmic localization of AID in steady-state conditions. Blocking nuclear import alters the balance between these processes in favor of cytoplasmic retention, resulting in reduced isotype class switching.This
work was supported by the Canadian Institutes of Health Research (MOP 84543)
and a Canada Research Chair (to J.M.D.). A.O. was supported by a fellowship
from the Canadian Institutes of Health Research Cancer Training Program at the
IRCM. V.A.C. was supported in part by a Michel Saucier fellowship from the
Louis-Pasteur Canadian Fund through the University of Montreal
Editing the genome of chicken primordial germ cells to introduce alleles and study gene function
With continuing advances in genome sequencing technology, the chicken genome
assembly is now better annotated with improved accuracy to the level of single
nucleotide polymorphisms. Additionally, the genomes of other birds such as the duck,
turkey and zebra finch have now been sequenced. A great opportunity exists in avian
biology to use genome editing technology to introduce small and defined sequence
changes to create specific haplotypes in chicken to investigate gene regulatory
function, and also perform rapid and seamless transfer of specific alleles between
chicken breeds. The methods for performing such precise genome editing are well
established for mammalian species but are not readily applicable in birds due to
evolutionary differences in reproductive biology.
A significant leap forward to address this challenge in avian biology was the
development of long-term culture methods for chicken primordial germ cells (PGCs).
PGCs present a cell line in which to perform targeted genetic manipulations that will
be heritable. Chicken PGCs have been successfully targeted to generate genetically
modified chickens. However, genome editing to introduce small and defined sequence
changes has not been demonstrated in any avian species. To address this deficit, the
application of CRISPR/Cas9 and short oligonucleotide donors in chicken PGCs for
performing small and defined sequence changes was investigated in this thesis.
Specifically, homology-directed DNA repair (HDR) using oligonucleotide donors
along with wild-type CRISPR/Cas9 (SpCas9-WT) or high fidelity CRISPR/Cas9
(SpCas9-HF1) was investigated in cultured chicken PGCs. The results obtained
showed that small sequences changes ranging from a single to a few nucleotides could
be precisely edited in many loci in chicken PGCs. In comparison to SpCas9-WT,
SpCas9-HF1 increased the frequency of biallelic and single allele editing to generate
specific homozygous and heterozygous genotypes. This finding demonstrates the
utility of high fidelity CRISPR/Cas9 variants for performing sequence editing with
high efficiency in PGCs.
Since PGCs can be converted into pluripotent stem cells that can potentially
differentiate into many cell types from the three germ layers, genome editing of PGCs
can, therefore, be used to generate PGC-derived avian cell types with defined genetic
alterations to investigate the host-pathogen interactions of infectious avian diseases.
To investigate this possibility, the chicken ANP32A gene was investigated as a target
for genetic resistance to avian influenza virus in PGC-derived chicken cell lines.
Targeted modification of ANP32A was performed to generate clonal lines of genome-edited
PGCs. Avian influenza minigenome replication assays were subsequently
performed in the ANP32A-mutant PGC-derived cell lines. The results verified that
ANP32A function is crucial for the function of both avian virus polymerase and
human-adapted virus polymerase in chicken cells. Importantly, an asparagine to
isoleucine mutation at position 129 (N129I) in chicken ANP32A failed to support
avian influenza polymerase function. This genetic change can be introduced into
chickens and validated in virological studies. Importantly, the results of my
investigation demonstrate the potential to use genome editing of PGCs as an approach
to generate many types of unique cell models for the study of avian biology.
Genome editing of PGCs may also be applied to unravel the genes that control the
development of the avian germ cell lineage. In the mouse, gene targeting has been
extensively applied to generate loss-of-function mouse models to use the reverse
genetics approach to identify key genes that regulate the migration of specified PGCs
to the genital ridges. Avian PGCs express similar cytokine receptors as their
mammalian counterparts. However, the factors guiding the migration of avian PGCs
are largely unknown. To address this, CRISPR/Cas9 was used in this thesis to generate
clonal lines of chicken PGCs with loss-of-function deletions in the CXCR4 and c-Kit
genes which have been implicated in controlling mouse PGC migration. The results
showed that CXCR4-deficient PGCs are absent from the gonads whereas c-Kit-deficient
PGCs colonise the developing gonads in reduced numbers and are
significantly reduced or absent from older stages. This finding shows a conserved role
for CXCR4 and c-Kit signalling in chicken PGC development. Importantly, other
genes suspected to be involved in controlling the development of avian germ cells can
be investigated using this approach to increase our understanding of avian reproductive
biology.
Finally, the methods developed in this thesis for editing of the chicken genome may
be applied in other avian species once culture methods for the PGCs from these species
are develope
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